We plan to continue our detailed genetic analysis of Sindbis virus, an alphavirus. We have isolated numerous temperature sensitive mutants of the virus and partially characterized many of them. We have identified 7 cistrons, divided into 3 complementation groups of RNA plus mutants affecting the structural proteins and 4 groups of RNA minus mutants with defects in non-structural proteins. Although we intend to continue to look for particular classes of mutants, which are under-represented or not represented in our current catalog, (such as mutants in control functions, polymerase mutants, and mutants defective in protein processing), the primary goal of the present proposal is to study our present mutants on the molecular level. We plan to scan our mutants for the synthesis of non-infectious particles at the non-permissive temperature, and to look for altered E3 protein in the extracellular fluid. We plan to group the RNA plus or minus mutants (largely ignored so far) by complementation, hoping to find additional functional groups of non-structural proteins. We propose to study the forces responsible for the biological activity and stability of the nucleocapsid by a variety of techniques, and to compare core particles from HR and mutans at every step. We hope to sequence the N-terminal and C-terminal fragments of the structural proteins, to learn more about protein processing from precursor polypeptides. In particular, we want to study interesting regions of the polypeptides from mutants which fail to cleave under non-permissive conditions. We will continue to examine the molecular biology of alphavirus replication in arthropod cells. by studying the replication of temperature sensitive mutants in these cells, and comparing the mutant phenotypes shown in avian and arthropod hosts, and continuing the search for functions only required in the invertebrate host.